Rheumatoid arthritis (RA) is a chronic inflammatory disease of uncertain etiology. Since the cause is unknown, treatment has been directed at suppressing the signs and symptoms of chronic inflammation. Although many agents have been documented to decrease pain and swelling temporarily, none has been shown to have a major impact on the course of the disease. While therapeutic modalities have been developed for treatment of this disease.sup.1-4, uniform and persistent suppression of this condition has not been reported. Although current approaches remain promising, alternative means of drug development seem warranted and could yield not only new and effective treatment modalities, but also provide new insights into disease pathogenesis that could serve as the basis of future therapeutic innovations.
An area to search for new therapeutic interventions for different forms of arthritis, and particularly RA and other autoimmune diseases, is that of traditional Chinese medicines. One of these traditional medicines is from Tripterygium wilfordii Hook F, a shrub-like vine from the Celastraceae family.sup.5. Tripterygium wilfordii Hook F is known to contain a number of constituents, some of which appear to be toxic.sup.6. It is known that the leaves, stem, flowers, and the skin of the roots are poisonous and that ingestion can cause death.sup.7-9. In contrast, the woody portion of the roots of the plant is much less toxic. An extract of Tripterygium wilfordii Hook F prepared from the root of the plant, designated T.sub.2, has been described in the Chinese literature for the treatment of autoimmune diseases.sup.10-26. The preparation appeared to contain therapeutic components, and to have a reduced toxicity compared to other available preparations of the plant. The general impression has been that T.sub.2 is well-absorbed orally, appears to have at least acceptable toxicity compared with other available preparations of the plant, and as being effective in the treatment of various autoimmune diseases.
The T.sub.2 extract has been evaluated in a double-blind placebo controlled cross-over study involving 70 RA patients, these patients having had a mean duration of RA of 6 years.sup.10-11. Significant improvement in a variety of clinical parameters, particularly ESR, CRP, and Rheumatoid factor titers, was noted after 12 weeks of therapy in the experimental group compared with either baseline measurements or the placebo treated group. Of the patients treated, 82-93% noted improvement in different clinical criteria or laboratory correlates of inflammation. An immunosuppressive activity of T.sub.2 may be inferred from the finding that treatment induced inhibition of the production of IgM and IgM rheumatoid factor by the patients' peripheral blood mononuclear cells in vitro.sup.7. Toxicity, which consisted primarily of skin rash, gastrointestinal complaints and amenorrhea, was reportedly of a generally mild nature, and reversible with cessation of therapy.
The Chinese experience suggested that a daily dosage of 0.8-1.5 mg/kg of T.sub.2 was relatively safe and effective. Acute and chronic toxicity studies have been carried out with T.sub.2 in China using a variety of animal models. The LD.sub.50 of T.sub.2 in mice was found to be 159.7 .+-. 14.3 mg/kg.sup.27. The major chronic toxicity noted in rats administered 30 mg/kg for 90 days was azoospermia and decrease in testicular weight.sup.27. Lower dosages of T.sub.2 did not cause decreases in testicular weight. The toxicity studies, therefore, suggested that T.sub.2 exhibited a reasonable safety index and should be able to be administered to patients safely.
Research has begun in China to determine the spectrum of activity of various preparations of T. wilfordii. According to the reported results of these studies, extracts of TWH.F were able to inhibit E-rosette formation by guinea pig T cells, mitogen induced IL-2 production by mouse T cells and antigen stimulated migration of rat lymphocytes.sup.28,29. Components of T. wilfordii hook F known as triptonide and triptolide have been reported to inhibit the proliferation of lymph cells induced by concanavalin A.sup.30. Chloroform/methanol extracts of the plant, referred to as T.sub.2 in the literature, have been described as having significant activity in vivo against certain mouse leukemias and in vitro against cells derived from human carcinomas.sup.31. The capacity of T.sub.2 to suppress a number of animal models of autoimmune disease, including adjuvant arthritis and experimental allergic encephalomyelitis, has been reported.sup.28-29,32-36. Large concentrations of T.sub.2 preparations (30 mg/kg) have been reported to suppress delayed type hypersensitivity reactivity in mice and may also suppress graft versus host disease, as well as skin and heart allograft rejection.sup.6,32.
Studies on animal models of autoimmune disease has revealed that various preparations of T. wilfordii inhibited manifestations of immune and inflammatory responses in mice, guinea pigs and rats. A water extract of T. wilfordii has been examined for its activity in the MRL-lpr/lpr mouse, a model of generalized autoimmune disease which includes glomerulonephritis, vasculitis, arthritis and lymphadenopathy. Dramatic prolongation of survival and reduction of urinary protein were observed in MRL-lpr/lpr mice treated with the water extract at doses of 20 mg/kg three times a week.sup.33,37.
T.sub.2 -induced toxicity has also been examined in animal models.sup.38-41. The LD.sub.50 for acute toxicity in mice was 159.7 .+-. 14.3 mg/kg. Administration of T.sub.2 at 60 mg/kg for 60-80 days, which was higher than that used for the studies of efficacy, did not affect body weight or the histology of most visceral organs except that of the testes and thymus in mice. Long term treatment of dogs with less than 15 mg/kg of T.sub.2 for 14.5 months did not affect body weight or hepatic or renal function. No pathological changes of the testes of the treated animals were observed. However, treatment of female mice with T.sub.2 at a dosage equal to .about.21 mg/kg for 5 months reduced the frequency of pregnancies and the number of fetuses in pregnant mice. T.sub.2 treatment also decreased the activity of sperm of rats in a time dependent manner. Treatment of dogs for as long as 14.5 months with 10 mg/kg of T.sub.2 caused significant atrophy of the testes.
The T.sub.2 examined in the Chinese literature is a crude extract containing a mixture of materials, including various glycosides, alkaloids, and diterpenoids. The active principle, however, has not yet been identified. A few components have been purified, including triptolide, wilfordine, and related compounds, but no particular purified component which accounts for the therapeutic or immunosuppressive activity of T.sub.2 exists.sup.40. High concentrations of triptolide were reported to suppress B and T lymphocyte proliferation and interleukin-2 production by mouse spleen cells.sup.41. However, the concentrations of the T.sub.2 used were sufficiently high that significant nonspecific toxicity undoubtedly occurred.
In China, an ethyl acetate (EA) extract of T. wilfordii is manufactured by the Huang Shi Pharmaceutical Company of Hubei Province. This material is extracted from the root with the root skin present, and is known to contain multiple components including terpenes, alkaloids and glycosides. The active ingredient(s) of this preparation, however, is unclear. Some investigators postulate that the diterpene lactone, triptolide and its related compounds account for the therapeutic effect of the EA extract.sup.42-43. Since triptolide is thought to be one of the most potent compounds and to account for much of the efficacy and toxicity of T. wilfordii, the content of tryptolide has been used to standardize the ethyl acetate extract of T. wilfordii in China. HPLC analysis carried out by the present inventors demonstrate that the average triptolide content of the Chinese EA extract from different batches manufactured by Huang Shi Pharmaceutical Company, such as those extracts described in Chen et al..sup.44 and Fang et al.sup.45, was 1.33 .mu.g per mg.
Acute toxicity testing in mice (Hubei white) indicated that the LD.sub.50 of the EA extract was 608-858 mg/kg. This varied with the source of the plant material used and the season of harvest. Examination of mice dying during the acute toxicity test of the EA extract (714-1400 mg/kg every 3 days, p.o.) showed that the most significant changes developed in lymphatic organs, including induction of atrophy of the thymus. Microscopic examination demonstrated changes that were especially noteworthy in the lymphatic system. These included decreased numbers of nodules and lymphocytes in lymph nodes, spleen and intestine. Mild weight loss and atrophy of the testes along with a decrease in the number and degeneration of spermatocytes were also found in these mice.sup.46.
The ethyl acetate extract produced in China, administered at doses of between 40-80 mg/kg/day, has been reported to inhibit adjuvant induced arthritis and cotton ball induced granuloma in rats.sup.47-49. This effect was comparable to that of cyclophosphamide or prednisone in the same animal models. At these doses, the EA extract also exerted immunosuppressive effects on both antibody production against sheep red blood cells (SRBC) and delayed skin hypersensitivity induced by dicloronitrobenzene (DNCB) in mice.sup.47.
Various other components have been identified in T. wilfordii Hook F including triptolide.sup.49, triptonolide, isoneotriptophenolide and wilforonide.sup.50. About 0.133% of the Chinese ethyl acetate extract has been identified as triptolide.sup.49. However, the biological activity of these components has not yet been determined. A need continues to exist for the identification and isolation of the highly therapeutically active components in T. wilfordii without the disadvantages associated with currently available preparations of the plant, such as in the T.sub.2 preparation. Further characterization of the potentially therapeutically useful components in the plant would provide clinically valuable treatment alternatives for autoimmune diseases, including rheumatoid arthritis, systemic lupus, erythematosus and psoriasis, as well as other forms of immunosuppression.
Thus, an object of the present invention is to provide less toxic yet biologically potent preparations of T. wilfordii Hook F, and isolated components thereof, than those currently available. Accomplishment of this object will provide a T. wilfordii-derived product that is more clinically acceptable with a reduced toxicity risk for use in humans. An additional object of the invention is to provide improved methods for treating immunosuppression, such as in the treatment of autoimmune disease, and particularly rheumatoid arthritis.